Highly efficient and safe genome editing by CRISPR-Cas12a using CRISPR RNA with a ribosyl-2′-O-methylated uridinylate-rich 3′-overhang in mouse zygotes |
| |
| Authors: | Dae-In Ha Jeong Mi Lee Nan-Ee Lee Daesik Kim Jeong-Heon Ko Yong-Sam Kim |
| |
| Affiliation: | 1.Genome Editing Research Center, KRIBB, Daejeon, 34141 Republic of Korea ;2.KRIBB School of Bioscience, Korea University of Science and Technology (UST), 34141 Daejeon, Republic of Korea ;3.GenKOre, Daejeon, 34141 Republic of Korea |
| |
| Abstract: | The CRISPR-Cas12a system has been developed to harness highly specific genome editing in eukaryotic cells. Given the relatively small sizes of Cas12a genes, the system has been suggested to be most applicable to gene therapy using AAV vector delivery. Previously, we reported that a U-rich crRNA enabled highly efficient genome editing by the CRISPR-Cas12a system in eukaryotic cells. In this study, we introduced methoxyl modifications at C2 in riboses in the U-rich 3′-overhang of crRNA. When mixed with Cas12a effector proteins, the ribosyl-2′-O-methylated (2-OM) U-rich crRNA enabled improvement of dsDNA digestibility. Moreover, the chemically modified U-rich crRNA achieved very safe and highly specific genome editing in murine zygotes. The engineered CRISPR-Cas12a system is expected to facilitate the generation of various animal models. Moreover, the engineered crRNA was evaluated to further improve a CRISPR genome editing toolset.Subject terms: Gene targeting, Genetic engineering |
| |
| Keywords: | |
|
|
